: The overall objective of this project is to understand the mechanisms by which DNA adenine methylase (DAM), leucine-responsive regulatory protein (Lrp), and PapI orchestrate the reversible switch between OFF and ON Pap pili expression states in uropathogenic Escherichia coli (UPEC). Since Pap pili are an essential virulence determinant of UPEC, this work has direct application to addressing the problem of urinary tract infections. PapI is required for translocation of Lrp between promoter-proximal pap sites 1-2-3 and distal sites 4-5-6. The methylation status of GATC sites within the central pap sites 2 and 5 controls binding of Lrp and PapI-Lrp. Binding of Lrp to promoter proximal sites represses pap transcription whereas binding of Lrp to distal sites initiates activation of pap transcription. The first aim of this proposal is to determine how DAM and PapI control binding of Lrp to sites 1-2-3 and 4-5-6. The hypothesis that PapI alters Lrp binding specificity, facilitating interaction with specific base- pairs present within sites 2 and 5 will be tested. The base-pair contacts between PapI-Lrp and sites 2 and 5 will be identified by missing contact, SELEX, and mutational analyses. The second aim is to identify amino acids of Lrp that play important roles in responsiveness to PapI and DNA methylation. This will be accomplished by isolation of lrp mutants with altered responses to PapI and DNA methylation and by a genetic suppressor approach using pap mutants isolated in Aim 1. Photo-crosslinking studies are proposed to directly identify amino acids within Lrp that interact with sites 2 and 5, and to determine how these interactions are altered by GATC site methylation. The third aim focuses on in vivo Pap switch dynamics. The hypothesis that DNA replication is required for Pap pili switching will be tested by monitoring pap pili gene expression by fluorescence activated cell sorting in synchronized cells following induction of PapI. Further analysis of the methylation states of the pap GATC sites following DNA replication will be done to determine the roles of DNA methylation in phase switching. Lastly, studies are proposed to determine how the phase switch is controlled in UPEC under normal physiologic conditions. The studies proposed here will provide a framework for understanding how DNA methylation patterns regulate pili gene expression since many other pili operons expressed by pathogenic bacteria share common regulatory features with pap. This work has wide clinical relevance since DAM is essential for the virulence of a number of pathogens including UPEC. Salmonella typhimurium, and Yersinia pseudotuberculosis Finally, the pap system is a paradigm for the study of epigenetic regulation in eukaryotes, in which DNA methylation regulates biological processes including imprinting, tumor formation, and gene silencing.